The effects of melatonin on bovine uniparental embryos developmentin vitroand the hormone secretion of COCs

Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS) levels in parthenogenetic and androgenetic embryos and cumulus—oocyte complexes (COCs) hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL), respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05). The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05). Melatonin supplemented significantly increased the maturation rate of oocytein vitro(P < 0.05). More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05). To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1andStAR) were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1,CYP19A1andStAR). In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocytein vitromaturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency ofin vitrodeveloped embryos.

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3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors

Biology ◽

10.3390/biology10111101 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1101

Author(s):

Daniela Bebbere ◽

Stefano Mario Nieddu ◽

Federica Ariu ◽

Davide Piras ◽

Sergio Ledda

Keyword(s):

Mammalian Species ◽

3D Culture ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Vitro Fertilization ◽

Beneficial Effects ◽

Fumed Silica Nanoparticles ◽

Liquid Marble

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus–oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans.

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346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab346 ◽

2007 ◽

Vol 19 (1) ◽

pp. 288

Author(s):

C. Kubota ◽

T. Kojima ◽

T. Nagai ◽

X. Tian ◽

X. Yang

Keyword(s):

Subsequent Development ◽

Industrial Applications ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Bovine Embryo ◽

Becton Dickinson ◽

In Vitro Storage ◽

Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco's PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus–oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL−1 polyvinyl alcohol (PVA). COCs were collected from follicles (3–8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5°C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 µg mL−1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281–286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS–IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM–IVF–IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student's t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM–IVF–IVC (bFF (n = 87): 0&percnt;, 0&percnt;; PBS/FBS (n = 72): 84&percnt;, 1&percnt;; and PBS/PVA (n = 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n = 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS–IVM–IVF (M199A/FBS (n = 97): 82&percnt;, 28&percnt;; and M199A/PVA (n = 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM–IVF–IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

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99 In Vitro Development of Alpaca Embryos Obtained by Bisection

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab99 ◽

2018 ◽

Vol 30 (1) ◽

pp. 189

Author(s):

L. Landeo ◽

R. S. Molina ◽

M. E. Zuñiga ◽

T. R. Gastelu ◽

C. Sotacuro ◽

...

Keyword(s):

Amino Acids ◽

Streptomyces Griseus ◽

Petri Dish ◽

Cumulus Cells ◽

Essential Amino Acids ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

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133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab133 ◽

2007 ◽

Vol 19 (1) ◽

pp. 184 ◽

Cited By ~ 3

Author(s):

T. Somfai ◽

M. Ozawa ◽

J. Noguchi ◽

H. Kaneko ◽

K. Ohnuma ◽

...

Keyword(s):

Polar Body ◽

Antioxidant Defense System ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Cleavage Rate ◽

Sperm Penetration ◽

Second Polar Body ◽

Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

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163IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab163 ◽

2004 ◽

Vol 16 (2) ◽

pp. 204 ◽

Cited By ~ 1

Author(s):

J. Ye ◽

K.H.S. Campbell ◽

M.R. Luck

Keyword(s):

Cumulus Cells ◽

Treated Group ◽

Blastocyst Stage ◽

Developmental Competence ◽

Meiotic Maturation ◽

In Vitro Production ◽

Maturation Medium ◽

Pre Treatment ◽

Vitro Production

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25mM HEPES and sodium bicarbonate, 3mM L-glutamine, 0.1% (w/v) BSA, 0.57mM cysteine, 10ngmL−1 EGF, 0.2μgmL−1 pLH, 100μmL−1 penicillin and 0.1mgmL−1 streptomycin) or in DM supplemented with 50ngmL−1 pFSH (DMF) and 5μgmL−1 CHX for 12h. COCs were then further cultured in the same DM without CHX for 24–30h or in DMF for 36h. For controls, COCs were cultured conventionally in DM for 42h or DMF for 48h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300000mL−1 for 6h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20μM adenosine and 0.2mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO2 in air and IVC in 5% CO2, 5% O2, 90% N2, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6±3.8%) was similar to that of controls (40.4±3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9±1.2%) than in controls (9.6±1.3%; P<0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7±5.0% and 22.3±2.4%, respectively; P<0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)

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Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes

Reproduction ◽

10.1530/rep-06-0288 ◽

2007 ◽

Vol 134 (3) ◽

pp. 405-414 ◽

Cited By ~ 28

Author(s):

Eugine Lee ◽

Yeon Ik Jeong ◽

Seon Mi Park ◽

Jong Yun Lee ◽

Ji Hye Kim ◽

...

Keyword(s):

Culture Media ◽

Polar Body ◽

Blastocyst Stage ◽

Developmental Competence ◽

Follicular Cells ◽

Beneficial Effects ◽

First Polar Body ◽

Cytoplasmic Maturation ◽

Neurotropic Factor

In an effort to improve the quality ofin vitroproduced porcine embryos, we investigated the effect of brain-derived neurotropic factor (BDNF), a neurotropin family member, onin vitromaturation (IVM) of porcine oocytes. The expression of BDNF and truncated isoforms of its receptor, tyrosine kinase B (TrkB), and p75 common neurotropin receptor was detected in both follicular cells and metaphase-I stage oocytes by RT-PCR. However, mRNA of full-length TrkB was not found in oocytes although it was detected in follicular cells. The expression pattern of BDNF and TrkB was confirmed by immunohistochemistry. Supplementation with BDNF (30 ng/ml) during IVM significantly (P< 0.05) increased the first polar body extrusion and glutathione levels in oocytes, whereas the effect of BDNF on nuclear maturation was diminished when gonadotropin and epidermal growth factor (EGF) were added to the culture media. However, treatment with BDNF (30 ng/ml) along with EGF (10 ng/ml) in the presence of gonadotropin significantly (P< 0.05) increased the developmental competence of oocytes to the blastocyst stage after bothin vitrofertilization (IVF; 29.1% when compared with control, 15.6%) and somatic cell nuclear transfer (SCNT; 13.6% when compared with control, 3%). This appeared to reflect a stimulatory interaction between BDNF and EGF to enhance the cytoplasmic maturation of oocytes to support successful preimplantation development. In conclusion, BDNFenhanced nuclearand cytoplasmic maturation of oocytes by autocrine and/or paracrine signals. Also, when used together with EGF, BDNF increased the developmental potency of embryos after IVF and SCNT, demonstrating an improvedin vitroproduction protocol for porcine oocytes.

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101 OVARIAN RESPONSE AND DEVELOPMENTAL COMPETENCE OF OOCYTES COLLECTED BY OPU IN SHEEP TREATED WITH GnRH ANTAGONIST

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab101 ◽

2005 ◽

Vol 17 (2) ◽

pp. 201

Author(s):

F. Berlinguer ◽

A. Gonzalez-Bulnes ◽

S. Succu ◽

G. Leoni ◽

I. Rosati ◽

...

Keyword(s):

Single Dose ◽

Gnrh Antagonist ◽

Ovarian Response ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Gnrh Antagonists ◽

Group A ◽

Group B

The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovulatory treatment protocols in sheep increases the number of smaller follicles able to grow and ovulate in response to the exogenous FSH treatment (Lopez-Alonso C et al. 2004 Reprod. Fertil. Dev. 16, 233). The aim of our study was to test if such treatment affects the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from GnRH-antagonist treated sheep during an ovarian by perstimulation protocol. Adult Sarda sheep (n = 18) were synchronized by the insertion of intravaginal sponges (Day 0) which were left in situ for 12 days; on Day 7, group A (n = 10) received a single dose of 3 mg of Antarelix (Teverelix, Europeptides, France) s.c., while group B (n = 8) served as control. All animals received 96 IU of FSH (Ovagen, ICP, New Zealand) administered in 4 equal doses given i.m. every 12 h starting on Day 10. Twelve hours after the last FSH administration oocytes were collected by OPU technique. Follicular growth was monitored by transrectal ultrasonography from Day 7 to Day 11. Collected oocytes were matured, fertilized, and cultured in vitro up to blastocyst stage under standard conditions used in our laboratory (Berlinguer F et al. 2004 Theriogenology 61, 1477–1486). After IVF, uncleaved oocytes were stained with acetolacmoid to evaluate chromatin configuration, while the cleaved ones were cultured in SOF + 0.4% BSA up to the blastocyst stage. Data were analyzed by ANOVA statistical analysis after arcsine transformation of the value percentages. Ultrasonographic monitoring showed a significant increase in the number of follicles (mean ± SEM) present in the ovaries from Day 8 to Day 11 of treatment in group A compared to group B (Day 8: 19 ± 5.1 vs. 13 ± 3.4, P > 0.05; Day 9: 20.1 ± 4.6 vs. 14.1 ± 2.4, P > 0.001; Day 10: 22.5 ± 6.1 vs. 14.7 ± 2.7, P > 0.001; Day 11: 25.3 ± 5.1 vs. 20.5 ± 4.1, P > 0.05), thus confirming that GnRH antagonist administration enhances ovarian response to exogenous FSH stimulation. On the other hand, oocytes collected from untreated sheep lead to a higher blastocyst output (P = 0.014), as illustrated in the table. These results indicated that although GnRH antagonist administration caused a significant increase in the ovarian response to the hormonal treatment, the final blastocyst output was significantly lower compared to that of the control group. This finding seems to suggest an impairment in the developmental competence of treated sheep oocytes. Table 1. In vitro maturation, fertilization, and developmental capacity of oocytes collected from follicles of GnRH antagonist-treated (group A) and untreated (group B) sheep This work was supported by funds from the Spanish MEC (projects SC 00-051-C3.1 and HI2002-0004) and the Italian MIUR (cofin).

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110 VITRIFICATION DEVICES AFFECT DEVELOPMENTAL COMPETENCE AND BIOCHEMICAL PROPERTIES OF IVM OVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab110 ◽

2006 ◽

Vol 18 (2) ◽

pp. 163 ◽

Cited By ~ 5

Author(s):

S. Succu ◽

G. G. Leoni ◽

F. Berlinguer ◽

F. Mossa ◽

M. Galioto ◽

...

Keyword(s):

Standard Condition ◽

Biochemical Properties ◽

Blastocyst Stage ◽

Developmental Competence ◽

Mitogen Activated Protein Kinase ◽

Control Group ◽

Parthenogenetic Activation ◽

Molecular Alterations ◽

Mapk Activity

This study was designed to evaluate the effects of 3 different vitrification devices on the developmental ability and on the maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of ovine oocytes in vitro-matured. Cumulus-oocytes complex (COCs) were in vitro-matured in TCM-199 supplemented with 10% FCS, 10 �L/mL of FSH/LH, and 1 �g/mL estradiol at 39�C and 5% CO2 atmosphere. For vitrification, oocytes were incubated in HEPES-buffered TCM-199 containing 20% FCS supplemented with DMSO (7.5%) and ethylene glycol (7.5%). After 3 min, oocytes were loaded into the same medium containing 0.5 m sucrose, 16.5% DMSO, and 16.5% EG, and then immersed into N2 using open pull straw (OPS; Vajta et al. 1998 Mol. Reprod. Dev.), cryoloop (CL; Lane et al. 1999 Nat. Biotechnol.), or cryotop (CT; Kuwayama and Kato 2000 J. Assist. Reprod. Genet.). After warming a part of oocytes were fertilized and cultured in vitro up to blastocyst stage in standard conditions (Leoni et al. 2005 Anim. Reprod. Sci., in press). The fertilization (23.8%, 31.6%, and 36.8% vs. 61.5%) and blastocyst rates (0, 12.5, and 0% vs. 50.0%) were lower for oocytes vitrified in OPS, CL, and CT, compared with control group. Control and vitrified IVM oocytes were valued for MPF and MAPK activity at 0 and 2 h after warming. In both post-warming experimental groups, the MAPK activity did not differ from control group. Immediately post-warming, MPF activity was lower in the vitrified groups compared with control oocytes (P < 0.01). If 100% is assigned to MPF activity in the control oocytes, those in the OPS, CL, and CT groups were, respectively, 63.3%, 65.5%, and 26.2%. After warming and culture for 2 h in standard condition, the activity of MPF was restored to values similar to control oocytes (87.0 and 76.8%, respectively) in the OPS and CL groups, whereas it was at the similar value in the CT group. To evaluate if the lowered MPF activity could cause parthenogenetic activation, the vitrified-warmed oocytes were cultured in SOF + 2% oestrus sheep serum in 5% O2, 5% CO2; after 27 h of culture, the oocytes were fixed and stained with propidium iodide to evaluate chromatin configuration. Results showed significantly higher parthenogenetic activation rates in the CT group compared with OPS and CL groups (54.5% vs. 22.6 and 27.4%, respectively). Our results indicate that the success of cryopreservation of the ovine oocyte is still very limited. The use of different vitrification devices not only modifies the ability to survive cryopreservation and developmental competence of oocytes but is also associated with important molecular alterations in the warmed oocyte cytoplasm. This work was supported by Cofin 2003.

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172 ADMINISTRATION OF GLUTATHIONE OR THIOREDOXIN TO MEDIUM REDUCES INTRACELLULAR REDOX STATUS AND IMPROVES EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE IVM/IVF OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab172 ◽

2006 ◽

Vol 18 (2) ◽

pp. 194

Author(s):

M. Ozawa ◽

T. Nagai ◽

M. Fahrudin ◽

N. W. K. Karja ◽

H. Kaneko ◽

...

Keyword(s):

Embryonic Development ◽

Culture Medium ◽

Redox Status ◽

Blastocyst Stage ◽

Developmental Competence ◽

Normal Embryo ◽

Control Group ◽

Intracellular Redox Status ◽

Intracellular Redox

Successful in vitro production of blastocysts from immature oocytes can be carried out using in vitro oocyte maturation (IVM), fertilization (IVF), and embryo culture (IVC) at a high level of repeatability in the porcine. However, the rates of in vitro development of IVM/IVF oocytes to the blastocyst stage remained around 20%. The environment in vitro is so simple and materially limited that there exist several stressors in vitro that disturb normal embryo development. Oxidative stress, which is caused by excess production of reactive oxygen species, is a major disturbing factor for the development of pre-implantation embryos in vitro. The series of present experiments were conducted using culture conditions with enhanced reducing capacity by the addition of glutathione (GSH) or thioredoxin to the culture medium to monitor developmental competence of porcine embryos and to verify their intracellular redox status. Cumulus-oocyte complexes were obtained from ovaries recovered from prepubertal gilts. Putative zygotes were produced by IVM of oocytes, followed by IVF (designated as Day 0). They were then cultured in modified NCSU-37 media containing GSH or thioredoxin as an antioxidant, or without any antioxidant (control), and blastocyst development rates on Day 6 were monitored. In addition, intracellular GSH content as a reducing parameter and intracellular H2O2 level as an oxidative parameter were measured; the intracellular redox status in the embryo was verified by the ratio of the GSH to the H2O2. Measurements in each group were replicated six times. Percentages of the embryos that developed to the blastocyst stage were significantly increased when 0.5 or 1.0 �M GSH (29.6 � 2.7% or 30.4 � 3.5%, and P < 0.05 or 0.01, respectively) or 1.0 mg/mL thioredoxin (30.6 � 2.4%, P < 0.01) was added to the medium compared to the percentage in the control group (20.1 � 2.2%). Intracellular redox status in embryos at the 8- to 12-cell stage or blastocysts was drastically reduced in GSH- or thioredoxin-added groups compared to that in the control group (P < 0.05 to 0.001). Furthermore, GSH or thioredoxin addition to the medium increased total cell numbers (48.3 � 2.1 to 49.2 � 2.1) and lowered ratios of apoptotic cells (6.2 � 0.6% to 7.0 � 0.7%) in blastocyst compared to those values in the control group (P < 0.05; cell number = 39.3 � 2.0, apoptosis rate = 11.1 � 1.1%) (37 to 53 embryos in each group were used for the TUNEL assay). These results suggest that the administration of GSH or thioredoxin to the culture medium improves in vitro embryonic development after IVM/IVF of oocytes, and that these beneficial effects are associated with maintenance of the intracellular redox status in a reduced state in porcine embryos.

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116 TAUROURSODEOXYCHOLIC ACID PREVENTS ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS IN PREIMPLANTATION PIG EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab116 ◽

2009 ◽

Vol 21 (1) ◽

pp. 158

Author(s):

J.-S. Kim ◽

K.-S. Lee ◽

B.-S. Song ◽

J.-Y. Zhang ◽

Y.-K. Choo ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Blastocyst Stage ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Control Group ◽

Tauroursodeoxycholic Acid ◽

Preimplantation Stage ◽

Induced Apoptosis

Apoptosis is an important determinant for the normal development of preimplantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. The efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting classic pathways of apoptosis. Therefore, we explored the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis in preimplantation porcine embryos. Also, TM (tunicamycin; an ER stress inducing chemical reagent) was used to investigate the effect of ER-stress on pig embryo development. After in vitro maturation and fertilization, presumptive porcine embryos were cultured in NCSU23 medium supplemented with 200 μ g mL–1 TUDCA or 1 μg mL–1 (TM) for 6 days at 39°C, 5% CO2 in air. All data were analyzed by using Duncan test of ANOVA by Statistical Analysis System (SAS). When treated with TM during culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage compared with 27.4% (28/102) of the embryos in the control group (P < 0.05). We also confirmed that TM stimulates up-regulation of ER stress response genes, such as XBP-1 mRNA, and induces a high rate of apoptosis. Whereas the frequency of blastocyst formation in the TUDCA-treated group was increased compared with that in the control group (32.8%, 49/149 v. 22.2%, 32/144), P < 0.05). Furthermore, the blastocyst cell number was enhanced (30.6 v. 39.5) and apoptosis reduced (TUNEL positive nuclei number, 6.0 v. 3.2) by TUDCA treatment in pig embryos. As the result of real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-xl gene was increased in the blastocyst stage by TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also confirmed that TUDCA decreases the rate of TM-induced apoptosis in preimplantation stage pig embryos. Our results indicate that TUDCA improves the developmental competence of porcine embryos by modulating the ER stress-induced apoptosis during preimplantation stage.

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